It is well recognized that decreased vascular endothelial growth factor A (VEGF-A) mRNA plays an important role in retinal vessel regression induced by hyperoxia. However, this concept has been challenged by increasing new evidence. Furthermore, VEGF-A strongly enhances Dll4 expression and inhibition of Dll4-Notch signaling leads to excessive sprouting angiogenesis. Recently, it is shown that inactivation of Dll4-Notch1 signaling reduce hyperoxia induced vessel regression. It is unknown whether sprouting angiogenesis contributes to the protective effect or not and further investigations are needed. Moreover, the expression of Dll4 or Notch1 activation in the regressing plexus remains elucidated. To determine the role of VEGF-A and Dll4-Notch1 signaling in hyperoxia induced vascular regression in the retina, we used mice at postnatal day 5 (P5) - P7. Hyperoxia induced massive vascular regression in the central plexus but not in the angiogenic plexus and had no effect on sprouting angiogenesis. Immunostaining showed that VEGF-A was significantly repressed in the angiogenic front region after hyperoxia exposure but not detectable in the central area of both normoxia and hyperoxia treated retinas. In contrast, Notch ligand Delta-like 4 (Dll4) and Notch1 intracellular domain (N1-ICD) expression were inhibited in the regressing capillaries of central retina but comparable in the angiogenic plexus after high oxygen treatment. Moreover, administration of Dll4 neutralizing antibody or γ-Secretase inhibitor DAPT significantly aggravated vessel regression induced by short-time hyperoxia administration. Our data show that repressed Dll4-Notch1 signaling pathway but not downregulation of VEGF-A expression are responsible for hyperoxia induced pervasive vessel regression.